DESCRIPTION (provided by applicant): Crossing-over is essential for accurate chromosome segregation during meiosis. In Saccharomyces cerevisiae, crossing-over is specifically promoted by ScZip3, a putative E3-ligase for SUMO. Allelic variants of the human ScZip3 homolog, RNF212, have been linked to changes in genome-wide meiotic recombination rates. To examine the role of mammalian ZIP3/RNF212, we have constructed a Zip3-/- knock-out mouse. Our long-term goal is to understand the roles of post-translational protein modification in meiotic crossing-over. The hypothesis is that ZIP3-promoted SUMOylation of recombination and/or chromosomal proteins promotes meiotic crossing-over. We will test this hypothesis using a combination of genetic, cytological and molecular approaches in both mouse and yeast. The Specific Aims are: 1. To analyze the function of mouse ZIP3. Preliminary immunofluorescence cytology shows that mouse ZIP3 (MmZIP3) localizes specifically to regions of chromosome synapsis. Localization can occur independently of recombination and is deregulated in the absence of synaptonemal complex protein, SYCP1. Immunofluorescence co-staining experiments will be used to test the idea that MmZIP3 normally localizes to sites of recombination. Additional mutant lines will be analyzed to further define the genetic requirements for MmZIP3 localization. Zip3-/- mutant mice will be analyzed in detail using histological and immunofluorescence cytology approaches. 2. To identify meiotic SUMO-conjugates and substrates of yeast and mouse ZIP3 proteins. The spectrum of SUMO-protein conjugates is dramatically altered in yeast zip3 mutants. ScZip3-dependent SUMO- conjugates will be identified by mass spectrometry. In parallel, candidate targets will be examined for ScZip3- dependent SUMOylation. In cases where yeast targets are conserved, these experiments will inform the identification of partners and potential substrates of MmZIP3. We will also utilize yeast 2-hybrid screening and co-immunoprecipitation from testis extracts to identify MmZIP3 partners and substrates. ScZip3- and MmZIP3- dependent in vitro SUMOylation assays will be reconstituted using purified components and used to confirm candidate substrates. 3. To analyze the role of SUMOylation for proteins identified in Specific Aim 2. Site-directed mutagenesis will be used to diminish SUMOylation of identified substrates in yeast and the effects on meiotic recombination and chromosome behavior will be examined using genetic analysis, specialized DNA physical assays, and immunofluorescence cytology. Relevance: Defects in recombination and post-translational modification have been linked to human infertility, miscarriage and genetic diseases, particularly cancer. An understanding of the mechanism and regulation of recombination will therefore help us better understand the etiology of these diseases. PUBLIC HEALTH RELEVANCE: Chromosome pairing and homologous recombination are required for sexual reproduction and chromosome repair. Defects in these processes are linked to human infertility, miscarriage and genetic diseases, particularly cancer. A greater understanding of their mechanism and regulation will help us better understand the etiology of these diseases.
|Effective start/end date||4/1/09 → 2/28/15|
- National Institutes of Health: $290,518.00
- National Institutes of Health: $294,755.00
- National Institutes of Health: $292,220.00
- National Institutes of Health: $289,760.00